Iron is essential for normal cellular functions in human body and the disturbances of iron homeostasis can cause life-threatening critical manifestations in various organs. Since very little iron is excreted, to prevent iron overload, the amount of iron in the body is tightly maintained by regulating intestinal iron absorption. Despite our advanced knowledge on the cellular utilization and storage of iron, the mechanism of iron transfer across the mucosa still remains unclear. The long-term goals of our research are to understand the cellular and molecular mechanism of the intestinal iron absorption processes and its regulation response to the various patho-physiological conditions. Recent discovery of two genes, hephaestin and ferroportinl (Iregl/MTP1), will help understand the mechanism of iron absorption and its regulation. While hephaestin was proposed to have an important role in iron transport across the basolateral membrane of mucosa, ferroportinl was expected to function as an iron exporter. The exact functions of these genes in the processes of intestinal iron absorption currently remain unknown. Thus, the primary goals of this exploratory R21 proposal are to understand and define functions of hephaestin and ferroportinl in the intestinal iron absorption processes. In this R21 project, we will utilize the human intestinal cell line to study the function of these two genes. We will transfect human intestinal Caco-2 cells with the genes encoding human hephaestin and ferroportinl to determine their roles in the processes of intestinal iron absorption. Specifically, these studies will: (1) characterize the cellular location and iron transport function of hephaestin and ferroportinl in human intestinal cell, and (2) determine the association of hephaestin with ferroportinl and how they might interact to affect the transfer of iron utilizing human intestinal Caco-2 cells overexpressing hephaestin but lacking ferroportinl (or hephaestin deficient but overexpressing ferroportinl). Results from this proposed study will provide critical information towards understanding the cellular and molecular mechanisms of regulating intestinal iron absorption. The completion of the proposed study in this R21 will be a critical step in achieving our long-term goals.